Biopterin analogs

ABSTRACT

This invention provides the intermediates of formula II-A below, useful to make the pterin compounds of formula I below. The pterin compounds are used in human and veterinary medicine.    &lt;IMAGE&gt;  IIA &lt;IMAGE&gt; I   wherein R represents a lower alkyl of 1 to 8 carbons.

This application is a continuation-in-part of application Ser. No.553,786, filed Sept. 19, 1983, now abandoned.

The present invention relates to a series of pteridines known as pterinswhich are analogs of tetrahydrobiopterin, to pharmaceutical formulationscontaining them, to processes for their preparation and to the usethereof in human medicine. More specifically the invention relates tocertain biopterin analogs and their use in the treatment of Parkinsonism(and other diseases caused by a deficiency of biogenicamines (e.g.,catecholamines and serotonin) in the brain and the perpheral nervoussystem) and the tetrahydrobiopterin-deficient phenylketonurias (atypicalPKU).

The invention accordingly provides the pterin compounds of formula (I)and their use in human and veterinary medicine. ##STR2## wherein Rrepresents a lower alkyl group (straight or branched) of 1-8 carbons(for this formula and all other formulas herein) including all stereoisomers thereof or a pharmaceutically acceptable salt thereof.

Most preferred of the compounds of formula (I) are(+-)-2-amino-5,6,7,8-tetrahydro-6-ethoxymethyl-4(3H)-pteridinone,(+-)-2-amino-5,6,7,8-tetra-hydro-6-methoxymethyl-4(3H)-pteridinone,(+-)-2-amino-5,6,7,8-tetrahydro-6-n-propoxymethyl-4(3H)-pteridinone and(+-)-2-amino-5,6,7,8-tetrahydro-6-n-butoxymethyl-4-(3H)-pteridinone orpharmaceutically acceptable salts thereof. It is generally accepted thatcertain substances known as neurotransmitters are required at themicroscopic regions, known as synapses, between nerve cells (neurons) totransmit the nerve impulses throughout the body.

Over thirty substances are known or suspected to be neurotransmittersand each has a characteristic excitatory or inhibitory effect onneurons. Excesses or deficiencies of these transmitters can bemanifested as moderate to severe neurological or mental disorders. Whileneurons are present throughout the body, imbalances of neurotransmittersat synapses of the neurons in the brain are by far the most critical andproduce the most pronounced effects.

Of the numerous neurotransmitters known or thought to be operating atsynapses, a smaller group collectively known as biogenic amines havereceived the most study. Particularly important members of this groupare the catecholamines such as dopamine and norepinephrine(noradrenaline) and the indole amine, serotonin.

Tetrahydrobiopterin (BH₄) is an essential cofactor for the rate limitingenzymes of biogenic amine synthesis, tyrosine and tryptophanhydroxylases, and for the liver enzyme which converts phenylalanine totyrosine (Kaufman, S. and Fisher, D. B., Molecular Mechanisms of OxygenActivation, Hayaishi, O. Ed., Acad. Press, N.Y. (1974)).

Knowledge of the chemical pathology of neurological disorders hasexpanded tremendously during the last two decades. For example,neurological disorders have been described whose symptoms can beassociated with decreases in the number of catecholamine and/orserotonin molecules released at certain synaptic sites. As a category,they may be thought of an `catecholamine-deficiency disorders`. Oneexample is Parkinson's disease (also known as Parkinsonism), where adeficiency in brain dopamine has been linked with the symptoms ofrigidity, tremor and akinesia. Another example is chronic preganglionicautonomic insufficiency known as the Shy-Dagger syndrome which isassociated with both peripheral sympathetic dysfunction and adegeneration of brain neurons in the basal ganglia. The peripheralsympathetic dysfunction most likely reflects a loss of formation andrelease of the pressor catecholamine norepinephrine, while the rigidityand alkinesia most likely reflects a loss of the capacity to formdopamine in certain brain regions.

In all of these cases the catecholamines, whose levels are diminished,are formed through the action of tyrosine hydroxylase which israte-limiting for their formation. This enzyme requires tyrosine, oxygenand a reduced pterin cofactor, tetrahydrobiopterin (BF₄), for activity.While oxygen and tyrosine are not normally limiting for tyrosinehydroxylase, the levels of BH₄ may normally be well below the levelsrequired to saturate this enzyme. In fact, there are reports in thescientific literature indicating the levels of this cofactor areseverely diminished in Parkinson's disease and in the Shy-Dragersyndrome (Nagatsu, T., Neurochem. Intern., 5, 27 (1983)). Logically, therate of dopamine biosynthesis would be increased by reversing thisdeficit. Administration of BH₄ has been shown to nearly double striataldopamine synthesis, as well as noradrenaline synthesis in peripheralnerves (Nagatsu, vide supra also Cloutier, G. and Weiner, N., J. Pharm.Exp. Ther., 186, 75 (1973)). In fact, BH₄ administration has beenreported to improve the symptoms of Parkinson's disease (Narabayashi,H., Kondo, T., Nagatsu, T., Sugimoto, T. and Matsurra, S., Proc. JapanAcad., 58, Ser. B, 283 (1982)). A group of physchiatric disorders knownas endogenous depression may also involve reduced neuronal formation ofcatecholamines and serotonin. In fact, BH₄ has recently been shown toimprove the symptoms of depression in several patients (Curtius, H., Muldner, H. and Niederwieser, A., J. Neurol. Transmission, 55, 301(1982)). This natural cofactor is however, expensive, unstable, and itpenetrates brain poorly. Thus, treatment with BH₄ is not the treatmentof choice. Previously known synthetic cofactor replacements for BH₄ allcause tyrosine hydroxylase (TH) to bind its substrate tyrosine moreweakly. Thus, the administration of these previously known syntheticcofactors for the acceleration of dopamine biosynthesis in the treatmentof catecholamine deficiency causes TH to become unsaturated withtyrosine.

The compounds of formula (I), quite unexpectedly, do not decrease thebinding of tyrosine to tyrosine hydroxylase and do promote dopamineformation by this enzyme at rates equal to that observed with thenatural cofactor, BH₄. Further, the compounds of formula (I) enter thebrain more easily than BH₄ and accelerate dopamine biosynthesis betterthan the natural cofactor when administered peripherally (orally). Thus,the administration of compounds of formula (I) for catecholaminedeficiency accelerate dopamine biosynthesis without coadministration ofexogenous tyrosine. An additional surprising property of the compoundsof formula (I) is selectivity for tyrosine hydroxylase when the carbonchain length of R is greater than two carbons. The compound of formula(I) wherein R=C₂ H₅ is active for tyrosine, tryptophan and phenylalaninehydroxylases, whereas the compounds of formula (I) where in R=C₃ H₇, C₄H₉ and C₈ H₁₇ are successively less active for tryptophan andphenylalanine hydroxylases. The greatest specificity for tyrosinehydroxylase among these compounds is found in that wherein R=C₄ H₉.

Endogenous (psychotic) depression is thought to involve decreased levelsof serotonin and noradrenaline at brain synapses. The compounds offormula (I) function as cofactors for tyrosine and tryptophanhydroxylases. Thus, these compounds will ameliorate the symptoms ofendogenous depression by promoting the synthesis of both catecholaminesand serotonin in the brain.

In addition to actions on the central nervous system, the compounds offormula (I) can replace BH₄ in the liver and promote the hydroxylationof phenylalanine. Indeed, a small percentage of all patients withphenylketonuria suffer from a tetrahydrobiopterin-deficient form ofphenylketonuria. This "atypical PKU" has been successfully treated withlarge quantities of BH₄ (Nagatsu, vide supra). Since the compounds offormula (I) are more lipophilic and have better bioavailability thanBH₄, considerably smaller doses could be used to treat this atypicalPKU. The compounds of formula (I) have at least 2 benefits. First, theyact as cofactors for hepatic phenylalanine hydroxylase to reduce plasmaphenylalanine levels. Second, they will act within the brain ascofactors for tyrosine and tryptophan hydroxylase, to correct the BH₄deficient reductions in cerebral catecholamine and serotonin levelswhich are also seen in atypical PKU. Paradoxically, large amounts ofphenylalanine inhibit phenylalanine hydroxylase with BH₄ as cofactor. Incontrast, the compounds of formula (I) do not show this substrateinhibition and would remain effective in the presence of largecirculating levels of phenylalanine.

Tetrahydropterins, upon reacting with tyrosine, tryptophan orphenylalanine hydroxylase, give rise to an unstable quininoidintermediate which is reduced back to the tetrahydro form bydihydropteridine reductase (DHPR). The compounds of formula (I) arerecycled by DHPR at rates three times faster than that of the naturalcofactor. This feature would extend the useful biologic life of thesecompounds, resulting in a favorable duration of action.

Tetrahydropterins, after enzymatic or chemical oxidation with molecularoxygen can also rapidly form the 7,8-dihydropterin structure which isnot reducible by DHPR. Dihydrofolate reductase (DHFR) will reducecertain 7,8-dihydropterins to the corresponding tetrahydropterins.7,8-Dihydrobiopterin (7,8-BH₂) can be reduced at a rate 2.5% that ofdihydrofolate, the natural substrate. Previously known TH cofactoranalogs are reduced at rates even slower than 7,8-BH₂ which shortenstheir lifetime in vivo. Surprisingly the dihydropterins derived fromcompounds of formula (I) are reduced by DHFR at rates 8 times, and more,faster than BH₂. Thus, DHFR increases the effective in vivo lifetime ofcompounds of formula (I) which improves their efficacy.

Compounds of formula (I) and their salts may be synthesized by methodsknown in the art of synthesis of compounds having analogous structures.In compounds of formula (IIA), (III), (V) and (VI) n=0 or 1.

A method of preparing compounds of formula (I) comprises reacting acompound of formula (IIA) with a reducing agent capable of donatinghydrogens such as H₂ /catalyst, sodium cyanoborohydride or sodiumborohydride all in a suitable solvent under conditions normally used forthese reagents. Compounds of formula (I) may also be prepared byreduction of compounds of formula (IIB), the 7,8-BH₂ compounds (videsupra), in vitro or in vivo with a suitable enzymatic reagent such asdihydrofolate reductase. ##STR3##

Compounds of formula (II) may be prepared by hydrolysis of a compound offormula (III) with a suitable agent such as an aqueous sodium hydroxidesolution. ##STR4##

In turn, compounds of formula (III) may be prepared by condensation ofthe compound of formula (IV) with compounds of formula (V) in thepresence of a base, such as sodium alkoxide, in an alcohol underconditions similar to those described in Taylor, et al., J. Org. Chem.,2817, 38 (1973). A specific example is sodium methoxide in methanol.##STR5##

Compounds of formula (V) may be prepared by refluxing a compound offormula (VI) in an alcohol of formula (VII). ##STR6##

Compounds of this invention may be used to treat Parkinson's disease,endogenous depression, orthostatic hypotension, muscular dystonia andother disorders which arise from deficiencies of catecholamines andserotonin at the pre-synaptic sites of neuronal junctions. Thesecompounds may also be used to treat the BH₄ -deficient phenylketonurias(atypical PKU).

The amount of the active compound, i.e. a compound of formula (I),required for use in the above disorder will, of course, vary with theroute of administration, the condition being treated, and the personundergoing treatment, but is ultimately at the discretion of thephysician. However, a suitable dose for treating these disorders is inthe range of from 0.5 to 20 mg per kilogram body weight per daypreferably from 1 to 10 mg/kg body weight per day, most preferably from2 to 7 mg/kg body weight per day, a typical preferred dose is 5 mg/kgbody weight per day.

The desired dose is preferably presented as between one and foursubdoses administered at appropriate intervals throughout the day. Thuswhere three sub-doses are employed each will lie in the range of from0.17 to 6.7 mg/kg body weight; a typical dose from a human recipientbeing 1.7 mg/kg body weight.

If desirable, the catecholamine and serotonin precursors 1-tyrosine or1-tryptophan may be administered concurrently with a compound of formula(I) at the rate of 25 mg/kg to 1000 mg/kg body weight. These amino acidsmay be given in the same pharmaceutical formulation, e.g., tablet orcapsule, as a compound of formula (I) or in a separate pharmaceuticalformulation.

While it is possible for the active compound or compounds to beadministered alone as the raw chemicals, it is preferable to present theactive compound or compounds as pharmaceutical formulations.Formulations of the present invention comprise a compound of formula (I)together with one or more pharmaceutically acceptable carriers thereofand optionally any other active therapeutic ingredients.

The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. The carrier may contain apreservative agent such as ascorbic acid.

The formulations include those suitable for oral, rectal or parenteral(including subcutaneous, intramuscular and intravenous) administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a finelydivided solid carrier and then, if necessary, shaping the product intothe desired formulations or packaging in a suitable container.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the activecompound(s); as a powder or granules; or a suspension in a non-aqueousliquid such as a syrup, an elixir, an emulsion or a draught. The activecompound(s) may also be presented as a bolus or depot formulation.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, the active compound being in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine, comprisinga mixture of the powdered active compound(s) with any suitable carrier.

Formulations for rectal administration may be presented as a suppositorywith a usual carrier such as cocoa butter.

Formulations suitable for parenteral administration can be made sterileand packed as dry solids in sealed sterile nitrogen-purged containers.At the time of parenteral administration a specified amount of sterilewater is added to the drug formulation and the resulting solution isadministered to the patient with a minimum of delay since the compoundsof formula (I) tend to be unstable in aqueous solution over a period oftime.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavouring agents, binders, surfaceactive agents, thickeners, lubricants, preservatives (includingantioxidants) and the like.

Compounds of the formula (I) are particularly useful in increasing thesynthesis of tyrosine, dopamine, norepinephrine and serotonin in mammalssuch as rats and humans. Such effects are produced by administrationpreferably orally or parenterally.

The following examples are provided by the way of illustration of thepresent invention and should in no way be construed as a limitationthereof.

EXAMPLE 1(+-)-2-Amino-5,6,7,8-tetrahydro-6-methoxymethyl-4(3H)-pteridinone

2-Amino-3-cyano-5-chloromethyl pyrazine (0.95 g) was reacted with alarge excess of dry methanol, under nitrogen, at reflux, for about 18hours. The resulting reaction mixture was filtered and the filtrate wastaken to dryness by evaporation. A yellow residue was collected andextracted twice with boiling methylene chloride. These extracts werefiltered and the solvent evaporated to give2-amino-3-cyano-5-methoxymethylpyrazine which was then treated with anexcess of guanidine in methanol according to the method of Taylor (J.Org. Chem., 2817, 38, (1973)). Most of the product of this reactionprecipitated and was collected by filtration. The filtrate was mixedwith silica gel, stripped of solvent by evaporation, and the residue wasplaced on a silica gel column. This column was eluted with methanol (5%)in ethylacetate to yield a second crop of product which was added tothat previously collected. The combined crops were recrystallized frommethanol to yield 2,4-diamino-6-methoxymethyl pteridine (mp 248°-251°).

The diaminopteridine, vide supra, was hydrolyzed in 1N NaOH at 70° C.for 3 hr. The resulting basic solution was then acidified with aceticacid, and the yellow product was filtered, washed with 95% ethanol andethyl ether, and dried under vacuum overnight to yield 0.5 g (83%) of2-amino-6-methoxymethyl-4(3H)-pteridinone as a yellow powder: UV (0.1NNaOH) λ_(max) 255 nm (22300), 277.5 nm sh (6200), 364 nm (7100); NMR: δ(d₆ -DMSO) 3.36 (s, OMe); 4.53 (s, CH₂ O); 7.32 (br s, NH₂); and 8.63(s, 7-H). Anal. calcd. for C₈ H₉ N₅ O₂.7/10H₂ O: C, 43.72; H, 4.77; N,31.86. Found: C, 43.77; H, 4.77; N, 31.63.

The pteridinone (0.5 g, 2.4×10⁻³ mol) previously prepared, vide supra,was hydrogenated under 1 atm of H₂ in 30 mL of distilled trifluoroaceticacid over 0.02 g of PtO₂ for an hour. The mixture was diluted with 25 mLof 6N HCl, filtered, and evaporated. The crude product, the tetrahydrocompound, was recrystallized twice from 6N HCl/acetonitrile to give2-amino-5,6,7,8-tetrahydro-6-methoxymethyl-4(3H)-pteridinone as a lightbrown solid (0.6 g, 85%): UV: (0.1N HCl) λ_(max) 215.5 nm (19800), 264nm (19900); IR: (KBr) 3300, 1665, 1570, 1540, 1470, 1365, 1300, 1150,1110, 1005, 955, and 750 cm⁻¹ ; NMR: δ (d₆ -DMSO) 3.29 (s,OMe); 3.4(m,CH's); 3.6 (br s, OCH₂); 8.0 (NH). Anal. calcd. for C₈ H₁₃ N₅O₂.2HCl.1/2H₂ O: C, 32.78; H, 5.50; N, 23.89; Cl, 24.19. Found: C,32.75; H, 5.54; N, 23.80; Cl, 24.21.

EXAMPLE 2(+-)-2-Amino-5,6,7,8-tetrahydro-6-n-butoxymethyl-4(3H)-pteridinone

This compound was made in a manner similar to that of the6-methoxymethyl title compound described in Example 1 (vide supra)except for the two following major exceptions: then-butoxymethylpyrazine was made by heating the chloromethylpyrazine (VI)in n-butanol instead of methanol, and the guanidine reaction was carriedout in heated n-butanol instead of refluxing methanol.

Spectra and microanalytical data of the three intermediates and finalproduct are the following:

2A. 2-Amino-3-cyano-5-n-butoxymethylpyrazine. NMR: δ (d₆ -DMSO) 0.89 (t,3H); 1.1-1.8 (m, 4H); 3.43 (t, 2H); 4.35 (s, 2H); 7.22 (br s, 2H); 8.25(s, 1H). IR: (CHCl₃) 3694, 3526, 3414, 2930, 2866, 2220 (CN), 1723,1612, 1463, 1377, 1275, 1088 cm⁻¹.

2B. 2,4-Diamino-6-n-butoxymethylpteridine. mp 237°-238° C. dec; NMR: δ(d₆ -DMSO) 0.87 (t, 3H); 1.0-1.7 (m, 4H); 3.51 (t, 2H); 4.58 (s, 2H);6.63 (br s, 2H); 7.59 (br s, 2H); 8.73 (s, 1H). Anal. Calcd. for C₁₁ H₁₆N₆ O: C, 53.21; H, 6.50; N, 33.85. Found: C, 53.02; H, 6.51; N, 33.75.

2C. 2-Amino-6-n-butoxymethyl-4(3H)-pteridinone. mp >300°, UV: (0.1NNaOH) λ_(max) 256 (27,400), 276.5 sh (11,300), 364.5 (7,200) nm. NMR: δ(d₆ -DMSO) 0.87 (t, 3H); 1.0-1.8 (m, 4H); 3.50 (t, 2H); 4.57 (s, 2H);6.97 (br s, 2H); 8.67 (s, 1H) (ring NH exchanged out due to large amountof H₂ O present). Anal. calcd. for C₁₁ H₁₅ N₅ O₂. 0.3H₂ O: C, 51.87; H,6.17; N, 27.50. Found: C, 51.80; H, 6.17; N, 27.55.

2D. (±)-2-Amino-5,6,7,8-tetrahydro-6-n-butoxymethyl-4(3H)-pteridinone.mp 211°-212° (dec). UV: (0.1N HCl) λ_(max) 265 (15,800) nm. NMR: δ (d₆-DMSO) 0.88 (t, 3H); 1.0-1.7 (m, 4H); 3.0-3.8 (m, 7H); 4.95, 6.85, 7.45,10.5 (br humps). Anal. Calcd. for C₁₁ H₁₉ N₅ O₂. 1.75HCl.H₂ O: C, 39.42;H, 6.84; N, 20.90; Cl, 18.52. Found: C, 39.40; H, 6.74; N, 21.06; Cl,18.61.

EXAMPLE 3(+-)-2-Amino-5,6,7,8-tetrahydro-6-methoxymethyl-4(3H)-pteridinone

3A. 2-Amino-6-methoxymethyl-4(3H)-pteridinone-8-oxide.2,4-Diamino-6-methoxymethylpteridine-8-oxide (E. C. Taylor and T.Kobayashi, J. Org. Chem., 2817, 38, (1973)) was treated with sodiumhydroxide in a manner analogous to that described in Example 1 to givethe title compound as a solid mp >250°. UV: (0.1N HCl) λ_(max)262(22600), 290 sh (4900), 388 (6100) nm; NMR: δ (TFA) 3.69 (s, OCH₃);4.89 (s, OCH₂); 8.95 (s, H-7). IR: (KBr) 1725, 1700, 1650, 1605, 1490,1480, 1390, 1360, 1325, 1235, 1155, 1110, 1065d, 1015d, 970 cm⁻¹. Anal.calcd: C, 43.05; H, 4.06; N, 31.38. Found: C, 42.79; H, 4.14; N, 31.21.

3B. (+-)-2-Amino-5,6,7,8-tetrahydro-6-methoxymethyl-4(3H)-pteridinone.The product from example 3A (1 g) was dissolved in trifluoroacetic acid(10 mL) and at one atmosphere pressure of hydrogen over platinum dioxidecatalyst. 6N hydrochloric acid (10 mL) was then added to the reactionmixture which was subsequently filtered to remove the catalyst andevaporated. The crude product was purified by treatment of a solutionwith activated charcoal and by recrystallization from 6N hydrochloricacid/acetonitrile to give a solid which was dried at approximately 50°C. in vacuo. The analytical and spectroscopic characteristics of theproduct were identical to those of the compound described in Example 1.

EXAMPLE 4(+-)-2-Amino-5,6,7,8-tetrahydro-6-n-butoxymethyl-4(3H)-pteridinone

4A. 2-Amino-3-cyano-5-n-butoxymethylpyrazine-1-oxide. This compound wasprepared by reacting 2-amino-3-cyano-5-chloromethylpyrazine-1-oxide withn-butanol under conditions analogous to those described in Example 1. mp58°-60°. NMR: δ (d⁶ -DMSO) 0.84 (t, 3H); 1.0-1.8 (m, 4H); 3.41 (t, 2H);4.31 (s, 2H); 7.86 (br s, 2H); 8.37 (s, 1H). Anal. calcd. for C₁₀ H₁₄ N₄O₂ : C, 54.04; H, 6.35; N, 25.21. Found: C, 53.96; H, 6.39; N, 25.17.

4B. 2,4-Diamino-6-n-butoxymethylpteridine-8-oxide. The product ofExample 4A was reacted with guanidine in methanol according to theprocedure described in Example 2 to give the title compound; mp >250°.UV: (0.1N HCl) λ_(max) 252.5 (30,900), 282 (10,700), 343.5 (sh) (6,200),356.5 (8,500), 369.5 (7,800) nm. NMR: δ (d⁶ -DMSO) 0.88 (t, 3H); 1.0-1.8(m, 4H); 3.51 (t, 2H); 4.49 (s, 2H); 7.01 (br s, 2H); 7.75 (br s, 2H);8.37 (s, 1H). IR: (KBr) 3700-2800, 1632, 1546, 1461, 1356, 1331, 1163,1053, 978 cm⁻¹. Anal. calcd. for C₁₁ H₁₆ N₆ O₂ : C, 49.99; H, 6.10; N,31.80. Found: C, 49.79; H, 6.11; N, 31.67.

4C. 2-Amino-6-n-butoxymethyl-4(3H)-pteridinone-8-oxide. The product ofExample 4B was hyrolyzed according to the produce of Example 3A to givethe title compound. UV: (0.1N NaOH) λ_(max) 261.5, 288 (sh), 390 nm.NMR: δ (d⁶ -DMSO) 0.89 (t, 3H); 1.0-1.8 (m, 4H); 3.48 (t, 2H); 4.45 (s,2H); 6.25 (br s, 2H); 8.19 (s, *1H).

4D. (+-)-2-Amino-5,6,7,8-tetrahydro-6-n-butoxymethyl-4(3H)-pteridinone.The product of Example 4C was treated according to the proceduredescribed in Example 3B to give the title compound, the analytical andspectral details of which are described in Example 2.

EXAMPLE 5(+-)-2-Amino-5,6,7,8-tetrahydro-6-ethoxymethyl-4(3H)-pteridinone

The compound was made in a manner similar to that of the 6-methoxymethyltitle compound described in Example 1 except the ethoxymethylpyrazinewas made by heating the chloromethylpyrazine (VI) in ethanol instead ofmethanol.

Spectra and microanalytical data of the three intermediates and finalproduct are the following:

5A. 2-Amino-3-cyano-5-ethoxymethylpyrazine. NMR: δ (d₆ -DMSO) 1.15 (t,3H); 3.50 (q, 2H); 4.35 (s, 2H); 7.23 (br s, 2H); 8.25 (s, 1H). IR:(CHCl₃) ˜3400, 2222 (CN), 1611, 1378, 1098 cm⁻¹. Anal. calcd. for C₈ H₁₀N₄ O: C, 53.92; H, 5.66; N, 31.44. Found: C, 53.81; N, 5.68; N, 31.41.

5B. 2,4-Diamino-6-ethoxymethylpteridine. mp 247°-248° (dec.). NMR: δ (d₆-DMSO) 1.17 (t, 3H); 3.57 (q, 2H); 4.58 (s, 2H); 6.62 (br s, 2H); 7.58(br s, 2H); 8.73 (s, 1H). IR: (KBr) 3327, 1630, 1440, 1356 cm⁻¹. Anal.calcd. for C₉ H₁₂ N₆ O: C, 49.08; H, 5.49; N, 38.16. Found: C, 48.81; H,5.53; N, 38.03.

5C. 2-Amino-6-ethoxymethyl-4(3H)-pteridinone. mp >300°. UV: (0.1N NaOH)(qualitative) λ_(max) 254, 277 (sh), 363 nm. NMR: δ (d₆ -DMSO) 1.14 (t,3H); 3.54 (q, 2H); 4.55 (s, 2H); 8.66 (br s, 2H); 8.66 (s, 1H); 11.3 (brs, 1H).

IR: (KBr) 1694 (C═O) cm⁻¹. Anal. calcd. for C₉ H₁₁ N₅ O₂ 0.58 H₂ O: C,46.66; H, 5.29; N, 30.23. Found: C, 46.65; H, 4.99; N, 30.27.

5D. (+-)-2-Amino-5,6,7,8-tetrahydro-6-ethoxymethyl-4(3H)-pteridinone. mp210.5°-217° (dec.). UV: (0.1N HCl) λ_(max) 265.5 (14200) nm. NMR: δ (d₆-DMSO) 1.12 (t, 3H); 3.49 (q overlapping m, 7H); 4.0-8.0 (br humps). IR:(KBr) 1660 (C═O) cm⁻¹. Anal. calcd. for C₉ H₁₅ N₅ O₂.2HCl.H₂ O: C,34.18, H, 6.06; N, 22.15; Cl, 22.43. Found: C, 34.29; H, 6.11; N, 22.14;Cl, 22.50.

EXAMPLE 6(+-)-2-Amino-5,6,7,8-tetrahydro-6-n-propoxymethyl-4(3H)-pteridinone.

This compound was made in a manner similar to that of the6-methoxymethyl title compound described in Example 1 except that then-propoxymethylpyrazine was made by heating the chloromethylpyrazine(VI) in n-propanol instead of methanol.

Spectra and microanalytical data of the three intermediates and finalproduct are the following:

6A. 2-Amino-3-cyano-5-n-propoxymethylpyrazine. mp 79°-80.5°. NMR: δ (d₆-DMSO) 0.88 (t, 3H); 1.2-1.9 (m, 2H); 3.40 (t, 2H); 4.36 (s, 2H); 7.23(br s, 2H); 8.25 (s, 1H). Anal. calcd. for C₉ H₁₂ N₄ O: C, 56.23; H,6.29; N, 29.15. Found: C, 56.01; H, 6.30; N, 29.14.

6B. 2,4-Diamino-6-n-propoxymethylpteridine. mp 239°-243° (dec.). NMR: δ(d₆ -DMSO) 0.89 (t, 3H); 1.57 (sextet, 2H); 3.47 (t, 2H); 4.59 (s, 2H);4.59 (s, 2H); 6.61 (br s, 2H); 7.57 (br s, 2); 8.73 (s, 1H). IR: (KBr)3430, 3304, 3132, 1624, 1434, 1353, 1187, 1077, 1064, 813 cm⁻¹. Anal.calcd. for C₁₀ H₁₄ N₆ O: C, 51.27; H, 6.02; N, 35.88. Found: C, 51.14;H, 6.04; N, 35.85.

6C. 2-Amino-6-n-propoxymethyl-4(3H)-pteridinone. mp >250°; UV: (0.1NNaOH) λ_(max) 255 (25000), 276.5 (sh) (6800), 364 (8000) nm. NMR: δ (d₆-DMSO) 0.88 (t, 3H); 1.57 (sextet, 2H); 3.47 (t, 2H); 4.58 (s, 2H); 6.90(br s, 2H); 8.69 (s, 1H); 11.3 (br s, 1H). IR: (KBr) 1693 (C═O) cm⁻¹.Anal. calcd. for C₁₀ H₁₃ N₅ O₂.0.20 H₂ O: C, 50.28; H, 5.66, N, 29.32.Found: C, 50.25; H, 5.63; N, 29.45.

6D. (+-)-2-Amino-5,6,7,8-tetrahydro-6-n-propoxymethyl-4(3H)-pteridinone.mp 194° (dec.). UV: (0.1N HCl) 265 (15100) nm. NMR: δ (d₆ -DMSO) 0.88(t, 3H); 1.56 (sextet, 2H); 2.9-4.0 (m, 7H); 8.2 (br hump). IR: (KBr)1662 (C═O) cm⁻¹. Anal. calcd. for C₁₀ H₁₇ H₅ O₂.2HCl.H₂ O: C, 36.37; H,6.41; N, 21.21; Cl, 21.47. Found: C, 36.28; H, 6.41; N, 21.18; Cl,21.46.

EXAMPLE 7(+-)-2-Amino-5,6,7,8-tetrahydro-6-isopropoxymethyl-4(3H)-pteridinone

This compound was made in a manner similar to that of the6-methoxymethyl title compound described in Example 1 except that theisopropoxymethylpyrazine was made by heating the chloromethylpyrazine(VI) in isopropanol instead of methanol.

Spectra and microanalytical data of the three intermediates and thefinal product are the following:

7A. 2-Amino-3-cyano-5-isopropoxymethylpyrazine. mp 92°-94°. NMR: δ (d₆-DMSO) 1.07 (d, 6H); 3.61 (septet, 1H); 4.34 (s, 2H); 7.19 (br s, 2H);8.25 (s, 1H). Anal. calcd. for C₉ H₁₂ N₄ O: C, 56.23; H, 6.29; N, 29.15.Found: C, 56.17; H, 6.30; N, 29.11.

7B. 2,4-Diamino-6-isopropoxymethylpteridine. mp 285°-286° (dec.). NMR: δ(d₆ -DMSO) 1.16 (d, 6H); 3.73 (septet, 1H); 4.58 (s, 2H); 6.60 (br s,2H); 7.56 (br s, 2H); 8.73 (s, 1H). IR: (KBr) 3436, 3312, 3146, 2970,1630, 1559, 1437, 1366, 1194, 1037, 819 cm⁻¹. Anal. calcd. for C₁₀ H₁₄N₆ O: C, 51.27; H, 6.02; N, 35.88. Found: C, 51.18; H, 6.06; N, 35.86.

7C. 2-Amino-6-isopropoxymethyl-4(3H)-pteridinone. mp >300°. UV: (0.1NNaOH) λ_(max) 254.5 (26000), 275.5 (sh) (7800), 363.5 (8200) nm. NMR: δ(d₆ -DMSO) 1.18 (d, 6H); 3.74 (septet, 1H); 4.59 (s, 2H); 6.86 (br s,2H); 8.70 (s, 1H); ˜11.3 (br s, 1H). IR: (KBr) 1700 (C═O) cm⁻¹. Anal.calcd. for C₁₀ H₁₃ N₅ O₂ : C, 51.05; H, 5.57; N, 29.77. Found: C, 51.09;H, 5.62; N, 29.71.

7D.(+-)-2-Amino-5,6,7,8-tetrahydro-6-isopropoxymethyl-4(3H)-pteridinone. mp210° (dec.). UV: (0.1N HCl λ_(max) 265 (14800) nm. NMR: δ (d₆ -DMSO)1.12 (d, 6H); 3.2-3.9 (m, 6H); 8.05 (br hump). IR: (KBr) 1669 (C═O)cm⁻¹. Anal. calcd. for C₁₀ H₁₇ N₅ O₂.2HCl.H₂ O: C, 36.37; H, 6.41; N,21.21; Cl, 21.47. Found: C, 36.47; H, 6.42; N, 21.19; Cl, 21.49.

EXAMPLE 8 BIOLOGICAL DATA

Tyrosine hydroxylase was partially purified through a 25-45% ammoniumsulfate fraction. Specific activity of this preparation withtetrahydrobiopterin was 4.0 nmole/mg protein (min). The enzyme wasassayed by a modification of the method of Nagatsu, T., Levitt, M., andUdenfriend, S. (Anal. Biochem. 9, 122, (1964)) where the Dowexchromatography step was replaced by a charcoal extraction.

Tryptophan hydroxylase was assayed according to a modification of themethod of Renson J., et al (Biochem. Biophys. Acta 25: 504 (1966)). Thepreparation of tryptophan hydroxylase was a crude 30,000xg supernatantwhich had been desalted on a Sephadex G-25 (Trade Name) column(Boadle-Biker, M. C., Biochem Pharmacol. 28: 2129 (1979). The specificactivity of this tryptophan hydroxylase, using BH₄ a cofactor, wasapproximately 100 pmol product per milligram protein per minute at 37°.

Phenylalanine hydroxylase was measured as described by Shiman, R. etal., (J. Biol. Chem. 254: 11300 (1979)) except that the product of thereaction (tyrosine) was measured fluorometrically using the method ofWaalkes, T. P. and Udenfriend, S. (J. Lab. Clin. Med. 50: 733 (1957).Phenylalanine hydroxylase was prepared from rat liver using hydrophobicchromatography (Shiman, R. et al. (J. Biol. Chem. 254: 11,300 (1979)).The specific activity of the enzyme, using BH₄ as the co-factor, was 1.0μmol/mg protein per minute at 37°.

Dihydrofolate reductase assay

These assays were carried out on the 7,8-dihydropterin analogs of thecompounds of formula I which were tetrahydropterins. The7,8-dihydropterins were obtained commercially (in the case ofdihydrobiopterin) or synthesized from the corresponding compound offormula I. Synthesis of 7,8-dihydropterins involved the combination of0.27 μmole of a compound of formula I, 20 μg of horseradish peroxidase,0.6 μmole of hydrogen peroxide in 0.1 ml of 0.5M potassium phosphatebuffer, pH 7.5. After 3 min at room temperature, 10 μg of bovine livercatalase was added. The production of the 7,8-dihydropterin was verifiedby U.V. spectroscopy.

The assay was carried out with bovine liver DHFR using a modification ofthe method of Bailey and Ayling (J. Biol. Chem., 253, 1598 (1978)) wherethe desired dihydropterin replaced dihydrobiopterin, and the HPLC systemused consisted of an anaerobic 0.1M acetic acid (with or without 10%acetonitrite) solvent on a 25 cm×4.6 mm Dupont TMS-Zorbax column. In ourhands, dihydrobiopterin is reduced at a rate 2.5% that of the naturalsubstrate dihydrofolate.

Dihydropteridine reductase was assayed by the method of Craine, et al.,(J. Biol. Chem., 247, 6082 (1972)).

                  TABLE I                                                         ______________________________________                                        BIOPTERIN COFACTOR ANALOGS                                                               Compound                                                           ______________________________________                                                     6-Methoxymethyl-                                                              tetrahydropterin                                                                           Tetrahydrobiopterin                                 ______________________________________                                        Tyrosine                                                                      hydroxylase                                                                   Km substrate (μM)                                                                       21.9         16.0                                                Km pterin (μM)                                                                           65          110                                                 Vmax (%)     124          100                                                 Phenylalanine                                                                 hydroxylase                                                                   Km substrate (μM)                                                                       111          123                                                 Km pterin (μM)                                                                          12.9         7.9                                                 Vmax (%)      79          100                                                 Tryptophan                                                                    hydroxylase                                                                   Km substrate (μM)                                                                        28           15                                                 Km pterin (μM)                                                                          103           82                                                 Vmax (%)      54          100                                                 ______________________________________                                                     (Quinonoid   (Quinonoid                                                       dihydro)     dihydro)                                            ______________________________________                                        Dihydropteridine                                                              reductase                                                                     Km pterin (μM)                                                                           7           1.8                                                 Vmax (%)     295          100                                                 ______________________________________                                                     (7,8-Dihydro)                                                                              7,8-Dihydro                                         ______________________________________                                        Dihydrofolate                                                                 reductase                                                                     V (%)        800          100                                                 ______________________________________                                    

EXAMPLE 9: PHARMACEUTICAL FORMULATIONS 9A. Capsule

    ______________________________________                                        Ingredient       Amount per capsule (mg)                                      ______________________________________                                        Compound (Ia)    325.0                                                        Ascorbic Acid    174.0                                                        Corn Starch      100        mg                                                Stearic Acid     27         mg                                                ______________________________________                                    

The finely ground active compound was mixed with the powdered excipientslactose, corn starch and stearic acid and packed into gelatin capsule.

9B. Tablet

    ______________________________________                                        Ingredient      Amount per tablet (mg)                                        ______________________________________                                        Compound I      325.0                                                         Ascorbic Acid   125.0                                                         Corn Starch     50.0                                                          Polyvinylpyrrolidone                                                                          3.0                                                           Stearic Acid    1.0                                                           Magnesium stearate                                                                            1.0                                                           ______________________________________                                    

The active compound was finely ground and intimately mixed with thepowdered excipients lactose, corn starch, polyvinylpyrrolidone,magnesium stearate and stearic acid. The formulation was then compressedto afford one tablet weighing 505 mg.

9C. Suppository

    ______________________________________                                        Ingredient           Amount per suppository                                   ______________________________________                                        Compound (Ia)        325.0     mg                                             Butylated hydroxy toluene (BHT)                                                                    25        mg                                             Cocoa Butter or Wecobee.sup.+  Base q.s.                                                           2.0       g                                              ______________________________________                                         .sup.+ Wecobee is the trade name of a hydrogenated carboxylic acid.      

We claim:
 1. The compound of the formula (IIA) ##STR7## where n is 1 and R represents a lower alkyl of 1 to 8 carbons or a salt thereof.
 2. A compound of claim 1 which is2-amino-6-methoxymethyl-4(3H)-pteridinone-8-oxide.
 3. A compound of claim 1 which is2-amino-6-n-butoxymethyl-4(3H)-pteridinone-8-oxide. 